Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

Abstract

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

A link between transcription fidelity and pausing in vivo

“…Decrease of replication fork progression was in fact observed in the triple mutant, and the three factors are known to prevent replication arrest during nutrient stress 45 . Furthermore, we observed similar morphological defects in a Δ greA mutant of S. pneumoniae , which does not have other secondary channel binding factors 30 . Of course, the severe growth phenotypes could also be due to the accumulation of distinct defects, and direct evidence is still missing.…”

GreA and GreB Enhance Expression of Escherichia coli RNA Polymerase Promoters in a Reconstituted Transcription–Translation System

“…In bacterial cells multiple proteins can increase RNAP activity, which are not present in the minimal PURE system. The transcription elongation factors GreA and GreB from E. coli increase overall transcription elongation rates and stimulate promoter escape in a subset of promoters by re-activating backtracked elongation complexes [19][20][21][22] . We added transcription elongation factors GreA and GreB to a PURE reaction containing EcRNAP with σ70 (holoenzyme) and a DNA template expressing EGFP under control of a consensus sequence E. coli σ70 promoter.…”

GreA and GreB enhanceEscherichia coliRNA polymerase transcription rate in a reconstituted transcription-translation system

“…In bacterial cells multiple proteins can increase RNAP activity, which are not present in the minimal PURE system. The transcription elongation factors GreA and GreB from E. coli increase overall transcription elongation rates and stimulate promoter escape in a subset of promoters by re-activating backtracked elongation complexes [19][20][21][22] . We added transcription elongation factors GreA and GreB to a PURE reaction containing EcRNAP with σ70 (holoenzyme) and a DNA template expressing EGFP under control of a consensus sequence E. coli σ70 promoter and found that both proteins increased transcription rates ( Figure 1).…”